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Transcription factors are essential regulators of various physiological and pathological processes. However, detecting transcription factor-DNA binding activities is often time-consuming and labor-intensive. Homogeneous biosensors that are compatible with mix-and-measure protocols have the potential to simplify the workflow for therapeutic screening and disease diagnostics. In this study, we apply a combined computational-experimental approach to investigate the design of a sticky-end probe biosensor, where the transcription factor-DNA complex stabilizes the fluorescence resonance energy transfer signal of the donor-acceptor pair. We design a sticky-end biosensor for the SOX9 transcription factor based on the consensus sequence and characterize its sensing performance. A systems biology model is also developed to investigate the reaction kinetics and optimize the operating conditions. Taken together, our study provides a conceptual framework for the design and optimization of sticky-end probe biosensors for homogeneous detection of transcription factor-DNA binding activity., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Pak Kin Wong reports financial support was provided by NSF., (Copyright © 2023. Published by Elsevier Inc.)

Double-stranded (ds) biosensors are homogeneous oligonucleotide probes for detection of nucleic acid sequences in biochemical assays and live cell imaging. Locked nucleic acid (LNA) modification can be incorporated in the biosensors to enhance the binding affinity, specificity, and resistance to nuclease degradation. However, LNA monomers in the quencher sequence can also prevent the target-fluorophore probe binding, which reduces the signal of the dsLNA biosensor. This study investigates the influence of LNA modification on dsLNA biosensors by altering the position and amount of LNA monomers present in the quencher sequence. We characterize the fluorophore-quencher interaction, target detection, and specificity of the biosensor in free solution and evaluate the performance of the dsLNA biosensor in 2D monolayers and 3D spheroids. The data indicate that a large amount of LNA monomers in the quencher sequence can enhance the specificity of the biosensor, but prevents effective target binding. Together, our results provide guidelines for improving the performance of dsLNA biosensors in nucleic acid detection and gene expression analysis in live cells.

Bladder cancer is an increasingly common malignancy, and muscle invasive bladder cancer is associated with particularly high rates of morbidity and mortality. The morphologic and molecular diversity of bladder cancer poses significant challenges in elucidating the invasion mechanisms responsible for disease progression. Furthermore, conventional invasion assays do not provide a physiological context for studying bladder cancer invasion within 3D microenvironments and have limited ability to capture the contribution of cellular phenotypic heterogeneity to disease progression. Here, we describe the development of a 3D microtumor invasion model suitable for the analysis of cellular phenotypic heterogeneity in cell lines and primary tumor cells from bladder cancer patients. This model incorporates a self-assembly approach for recapitulating features of bladder cancer invasion in 3D microenvironments and probing the invasive cell subpopulations. The gene expression profiles of invading microtumors were analyzed by incorporating a gold nanorod-locked nucleic acid biosensor. The incorporation of the single cell biosensor and transient gene knockdown into the system revealed the formation of invasive leader cells with upregulated Delta-like ligand 4 (DLL4) expression as well as the role of NOTCH1-DLL4 signaling in collective bladder cancer invasion. The involvement of DLL4 expressing cells in bladder cancer invasion was also observed in patient samples obtained from transurethral resection. Collectively, our study demonstrates a 3D microtumor invasion model for investigating intracellular heterogeneity of bladder cancer invasion and analyzing patient derived samples toward personalized medicine applications.

The functions of RNA are tightly regulated via diverse intracellular mechanisms. However, probing the complex dynamics of endogenous RNA in live cells is a challenging task. In the present study, a DNA transformer is designed for visualizing the abundance, distribution, and mobility of endogenous mRNAs in live human cells. The transformable tetrahedral DNA (T-TED) probe has a flexible hinge structure and is programmed to conform into a 3D tetrahedron upon binding with the target mRNA. By incorporating Förster resonance energy transfer (FRET) imaging, super-resolution localization, and single particle tracking, the T-TED biosensor is applied for investigating the dynamics of Delta-like ligand 4 (Dll4) mRNA, which encodes a transmembrane protein, in human pulmonary microvascular endothelial cells. The data reveal unprecedented subpopulations of Dll4 mRNA with distinct mobility organized spatially in association with the endoplasmic reticulum and microtubule networks. The ability to monitor the dynamics of endogenous RNA in live human cells will provide a useful tool for studying the functions and regulation of RNA.

Nuclear factor kappa B (NF-κB), regulating the expression of several genes that mediate the inflammatory responses and cell proliferation, is one of the therapeutic targets for chronic inflammatory disease and cancer. A novel molecular binding scheme for the detection of NF-κB was investigated for its affinity to Ig-κB DNA composed by dye and quencher fluorophores, and this specificity is confirmed by competing with the DNA sequence that is complementary to the Ig-κB DNA. We create a normalization equation to remove the negative effects from the various initial fluorophore concentrations and the background noise. We also found that a periodic shaking at a frequency could help to stabilize the DNA⁻protein binding. The calibration experiment, using purified p50 (NF-κB), shows that this molecular probe biosensor has a detection limit on the order of nanomolar. The limit of detection is determined by the binding performance of dye and quencher oligonucleotides, and only a small portion of probes are stabilized by DNA-binding protein NF-κB. The specificity experiment also shows that p50/p65 heterodimer has the highest affinity for Ig-κB DNA; p65 homodimer binds with intermediate affinity, whereas p50 shows the lowest binding affinity, and Ig-κB DNA is not sensitive to BSA (bovine albumin serum). The experiment of HeLa nuclear extract shows that TNF-α stimulated HeLa nuclear extract has higher affinity to Ig-κB DNA than non-TNF-stimulated HeLa nuclear extract (4-h serum response). Therefore, the molecular binding scheme provides a rapid, quantitative, high throughput, and automated measurement of the DNA-binding protein NF-κB at low cost, which is beneficial for automated drug screening systems.

Amperometric biosensors are widely applied for rapid biomarker detection in physiological and environmental samples. The dynamics and linearity of the current signal, however, are only partially understood. This study investigates the diffusion-reaction kinetics of amperometric biosensing using a self-assembled monolayer (SAM) based biosensor for bacterial 16S rRNA. A numerical model is developed to optimize the chamber dimensions and elucidate the concentration dependences of the biosensor. The results revealed that depletion of substrates associated with the chamber dimension can limit the current signal in a target concentration dependent manner. This study provides practical guidelines in the design and interpretation of microfluidic amperometric biosensors for biochemical applications.

Transcription and translation are under tight spatiotemporal regulation among cells to coordinate multicellular organization. Methods that allow massively parallel detection of gene expression dynamics at the single cell level are required for elucidating the complex regulatory mechanisms. Here we present a multiplex nanobiosensor for real-time monitoring of protein and mRNA expression dynamics in live cells based on gapmer aptamers and complementary locked nucleic acid probes. Using the multiplex nanobiosensor, we quantified spatiotemporal dynamics of vascular endothelial growth factor A mRNA and protein expressions in single human endothelial cells during microvascular self-organization. Our results revealed distinct gene regulatory processes in the heterogeneous cell subpopulations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Bloodstream infections are major contributing factors of morbidity and mortality among children. Precise and timely identification of causative agents can improve the clinical management and outcome of the infection, potentially saving lives. Electrochemical biosensors previously described by Gao et al. (2017) have the potential to deliver greater speed and discrimination. However, to date there are no data that determine whether the age of the host would cause bacteria to demonstrate different growth characteristics, or whether pediatric samples would behave differently using this electrochemical biosensor. The importance of this knowledge gap is clear: the preclinical testing phase of this line of research is limited by the relative lack of pediatric healthy blood volunteers to complete this work. Therefore, in this study we have applied this novel technology to diagnose bacteria spiked into pediatric blood and compared directly with adult blood samples. Only 180 µL of blood was utilized from both adult and pediatric volunteers and inoculated with Escherichia coli 67, and the signals generated at different time points were compared. We were able to demonstrate that the signals generated by adult and pediatric blood were not significantly different with this detection technology.

Accurate and timely detection of bacterial pathogens will improve the clinical management of infections. Herein, we demonstrate an electrochemical biosensor that directly detects bacteria in human blood samples, resulting in the rapid diagnosis of a bloodstream infection. The multiplex biosensor detects the species-specific sequences of the 16S ribosomal RNA of bacteria for pathogen identification in physiological samples without preamplification. The analytical performance characteristics of the biosensor, including the limit of detection and probe cross-reactivity, are evaluated systematically. The feasibility of the biosensor for a diagnosis of a bloodstream infection is demonstrated by identifying bacterial clinical isolates spiked in whole blood and blood culture samples that were tested positive for bacteria. The electrochemical biosensor correctly identifies all the species in the samples with 100% concordance to microbiological analysis.

Background: Standard diagnosis of urinary tract infection (UTI) via urine culture for pathogen identification (ID) and antimicrobial susceptibility testing (AST) takes 2-3 d. This delay results in empiric treatment and contributes to the misuse of antibiotics and the rise of resistant pathogens. A rapid diagnostic test for UTI may improve patient care and antibiotic stewardship., Objective: To develop and validate an integrated biosensor assay for UTI diagnosis, including pathogen ID and AST, with determination of the minimum inhibitory concentration (MIC) for ciprofloxacin., Design, Setting, and Participants: Urine samples positive for Enterobacteriaceae (n=84) or culture-negative (n=23) were obtained from the Stanford Clinical Microbiology Laboratory between November 2013 and September 2014. Each sample was diluted and cultured for 5h with and without ciprofloxacin, followed by quantitative detection of bacterial 16S rRNA using a single electrochemical biosensor array functionalized with a panel of complementary DNA probes. Pathogen ID was determined using universal bacterial, Enterobacteriaceae (EB), and pathogen-specific probes. Phenotypic AST with ciprofloxacin MIC was determined using an EB probe to measure 16S rRNA levels as a function of bacterial growth., Measurements: Electrochemical signals for pathogen ID at 6 SD over background were considered positive. An MIC signal of 0.4 log units lower than the no-antibiotic control indicated sensitivity. Results were compared to clinical microbiology reports., Results and Limitations: For pathogen ID, the assay had 98.5% sensitivity, 96.6% specificity, 93.0% positive predictive value, and 99.3% negative predictive value. For ciprofloxacin MIC the categorical and essential agreement was 97.6%. Further automation, testing of additional pathogens and antibiotics, and a full prospective study will be necessary for translation to clinical use., Conclusions: The integrated biosensor platform achieved microbiological results including MIC comparable to standard culture in a significantly shorter assay time. Further assay automation will allow clinical translation for rapid molecular diagnosis of UTI., Patient Summary: We have developed and validated a biosensor test for rapid diagnosis of urinary tract infections. Clinical translation of this device has the potential to significantly expedite and improve treatment of urinary tract infections., (Published by Elsevier B.V.)

Cancer is a leading cause of death worldwide and metastases are responsible for over 90% of human cancer deaths. There is an urgent need to develop novel therapeutics for suppressing cancer invasion, the initial step of metastasis. Nevertheless, the regulation of cancer invasion is poorly understood due to a paucity of tools for monitoring the invasion process in 3D microenvironments. Here, we report a double-stranded locked nucleic acid (dsLNA) biosensor for investigating 3D collective cancer invasion. By incorporating multiphoton microscopy and the dsLNA biosensor, we perform dynamic single cell gene expression analysis while simultaneously characterizing the biomechanical interaction between the invading sprouts and the extracellular matrix. Gene profiling of invasive leader cells and detached cells suggest distinctive signaling mechanisms involved in collective and individual invasion in the 3D microenvironment. Our results underscore the involvement of Notch signaling in 3D collective cancer invasion, which warrants further investigation toward antimetastasis therapy in the future.

This study reports the development of a portable whole cell biosensor system for environmental monitoring applications, such as air quality control, water pollution monitoring, and radiation leakage detection. The system consists of a lightweight mechanical housing, a temperature regulating system, and a microfluidic bacterial inoculation channel. The overall system, which is less than 200 g, serves as a portable incubator for cell inoculation and can be mounted on an unmanned aerial vehicle for monitoring remote and unreachable locations. The feedback control system maintains the inoculation temperature within 0.05 °C. The large surface-to-volume ratio of the polydimethylsiloxane microchannel facilitates effective gas exchange for rapid bacterial growth. Molecular dynamic simulation shows effective diffusion of major gas pollutants in PDMS toward gas sensing applications. By optimizing the design, we demonstrate the operation of the system in ambient temperatures from 5 °C to 32 °C and rapid bacterial growth in microchannels compared to standard bacterial culture techniques.

A gold nanorod-locked nucleic acid nano-biosensor for dynamic single-cell gene expression analysis in living cells and tissues is developed. The nanoparticle facilitates endocytic delivery and dynamic monitoring of the gene expression in human umbilical cord endothelial cells, mouse skin tissues, mouse retina tissues, and mouse cornea tissues at the single-cell level., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rapid pathogen detection and antimicrobial susceptibility testing (AST) are required in diagnosis of acute bacterial infections to determine the appropriate antibiotic treatment. Molecular approaches for AST are often based on the detection of known antibiotic resistance genes. Phenotypic culture analysis requires several days from sample collection to result reporting. Toward rapid diagnosis of bacterial infection in non-traditional healthcare settings, we have developed a rapid AST approach that combines phenotypic culture of bacterial pathogens in physiological samples and electrochemical sensing of bacterial 16S rRNA. The assay determines the susceptibility of pathogens by detecting bacterial growth under various antibiotic conditions. AC electrokinetic fluid motion and Joule heating induced temperature elevation are optimized to enhance the sensor signal and minimize the matrix effect, which improve the overall sensitivity of the assay. The electrokinetics enhanced biosensor directly detects the bacterial pathogens in blood culture without prior purification. Rapid determination of the antibiotic resistance profile of Escherichia coli clinical isolates is demonstrated.

Rapid diagnosis of infectious diseases and timely initiation of appropriate treatment are critical determinants that promote optimal clinical outcomes and general public health. Conventional in vitro diagnostics for infectious diseases are time-consuming and require centralized laboratories, experienced personnel and bulky equipment. Recent advances in biosensor technologies have potential to deliver point-of-care diagnostics that match or surpass conventional standards in regards to time, accuracy and cost. Broadly classified as either label-free or labeled, modern biosensors exploit micro- and nanofabrication technologies and diverse sensing strategies including optical, electrical and mechanical transducers. Despite clinical need, translation of biosensors from research laboratories to clinical applications has remained limited to a few notable examples, such as the glucose sensor. Challenges to be overcome include sample preparation, matrix effects and system integration. We review the advances of biosensors for infectious disease diagnostics and discuss the critical challenges that need to be overcome in order to implement integrated diagnostic biosensors in real world settings.

Rapid detection of bacterial pathogens is critical toward judicious management of infectious diseases. Herein, we demonstrate an in situ electrokinetic stringency control approach for a self-assembled monolayer-based electrochemical biosensor toward urinary tract infection diagnosis. The in situ electrokinetic stringency control technique generates Joule heating induced temperature rise and electrothermal fluid motion directly on the sensor to improve its performance for detecting bacterial 16S rRNA, a phylogenetic biomarker. The dependence of the hybridization efficiency reveals that in situ electrokinetic stringency control is capable of discriminating single-base mismatches. With electrokinetic stringency control, the background noise due to the matrix effects of clinical urine samples can be reduced by 60%. The applicability of the system is demonstrated by multiplex detection of three uropathogenic clinical isolates with similar 16S rRNA sequences. The results demonstrate that electrokinetic stringency control can significantly improve the signal-to-noise ratio of the biosensor for multiplex urinary tract infection diagnosis., From the Clinical Editor: Urinary tract infections remain a significant cause of mortality and morbidity as secondary conditions often related to chronic diseases or to immunosuppression. Rapid and sensitive identification of the causative organisms is critical in the appropriate management of this condition. These investigators demonstrate an in situ electrokinetic stringency control approach for a self-assembled monolayer-based electrochemical biosensor toward urinary tract infection diagnosis, establishing that such an approach significantly improves the biosensor's signal-to-noise ratio., (© 2013.)

To develop a portable point-of-care system based on biosensors for common infectious diseases such as urinary tract infection, the sensing process needs to be implemented within an enclosed fluidic system. On chip sample preparation of clinical samples remains a significant obstacle to achieving robust sensor performance. Herein AC electrokinetics is applied in an electrochemical biosensor cassette to enhance molecular convection and hybridization efficiency through electrokinetics induced fluid motion and Joule heating induced temperature elevation. Using E. coli as an exemplary pathogen, we determined the optimal electrokinetic parameters for detecting bacterial 16S rRNA in the biosensor cassette based on the current output, signal-to-noise ratio, and limit of detection. In addition, a panel of six probe sets targeting common uropathogenic bacteria was demonstrated. The optimized parameters were also validated using patient-derived clinical urine samples. The effectiveness of electrokinetics for on chip sample preparation will facilitate the implementation of point-of-care diagnosis of urinary tract infection in the future.

Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. The standard culture-based diagnosis of UTI has a typical delay of two to three days. In the absence of definitive microbiological diagnosis at the point of care, physicians frequently initiate empirical broad-spectrum antibiotic treatment, and this has contributed to the emergence of resistant pathogens. Biosensors are emerging as a powerful diagnostic platform for infectious diseases. Paralleling how blood glucose sensors revolutionized the management of diabetes, and how pregnancy tests are now conducted in the home, biosensors are poised to improve UTI diagnosis significantly. Biosensors are amenable to integration with microfluidic technology for point-of-care (POC) applications. This review focuses on promising biosensor technology for UTI diagnosis, including pathogen identification and antimicrobial susceptibility testing, and hurdles to be surpassed in the translation of biosensor technology from bench to bedside., (Published by Elsevier Ltd.)

Purpose: A significant barrier to efficient antibiotic management of infection is that the standard diagnostic methodologies do not provide results at the point of care. The delays between sample collection and bacterial culture and antibiotic susceptibility reporting have led to empirical use of antibiotics, contributing to the emergence of drug resistant pathogens. As a key step toward the development of a point of care device for determining the antibiotic susceptibility of urinary tract pathogens, we report on a biosensor based antimicrobial susceptibility test., Materials and Methods: For assay development bacteria were cultured with or without antibiotics, and growth was quantitated by determining viable counts and electrochemical biosensor measurement of bacterial 16S rRNA. To determine antibiotic susceptibility directly from patient samples, urine was cultured on antibiotic plates for 2.5 hours and growth was determined by electrochemical measurement of bacterial 16S rRNA. For assay validation 252 urine samples were collected from patients at the Spinal Cord Injury Service at Veterans Affairs Palo Alto Health Care System. The biosensor based antimicrobial susceptibility test was completed for samples containing gram-negative organisms. Pathogen identification and antibiotic susceptibility results were compared between our assay and standard microbiological analysis., Results: A direct biosensor quantitation of bacterial 16S rRNA can be used to monitor bacterial growth for a biosensor based antimicrobial susceptibility test. Clinical validation of a biosensor based antimicrobial susceptibility test with patient urine samples demonstrated that this test was 94% accurate in 368 pathogen-antibiotic tests compared to standard microbiological analysis., Conclusions: This biosensor based antimicrobial susceptibility test, in concert with our previously described pathogen identification assay, can provide culture and susceptibility information directly from a urine sample within 3.5 hours., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Urinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management., Methodology/principal Findings: The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both)., Conclusion/significance: We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for multiplexed detection of nucleic acid and protein as the next generation of urinary tract infection diagnostics.

Urine is the most abundant and easily accessible of all body fluids and provides an ideal route for non-invasive diagnosis of human diseases, particularly of the urinary tract. Electrochemical biosensors are well suited for urinary diagnostics due to their excellent sensitivity, low-cost, and ability to detect a wide variety of target molecules including nucleic acids and protein biomarkers. We report the development of an electrochemical immunosensor for direct detection of the urinary tract infection (UTI) biomarker lactoferrin from infected clinical samples. An electrochemical biosensor array with alkanethiolate self-assembled monolayer (SAM) was used. Electrochemical impedance spectroscopy was used to characterize the mixed SAM, consisted of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol. A sandwich amperometric immunoassay was developed for detection of lactoferrin from urine, with a detection limit of 145 pg/ml. We validated lactoferrin as a biomarker of pyuria (presence of white blood cells in urine), an important hallmark of UTI, in 111 patient-derived urine samples. Finally, we demonstrated multiplex detection of urinary pathogens and lactoferrin through simultaneous detection of bacterial nucleic acid (16S rRNA) and host immune response protein (lactoferrin) on a single sensor array. Our results represent first integrated sensor platform capable of quantitative pathogen identification and measurement of host immune response, potentially providing clinical diagnosis that is not only more expeditious but also more informative than the current standard., (Published by Elsevier B.V.)